All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.
Precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes. Gene expression involves a number of specific protein-protein interactions. Transcription of DNA into RNA involves a transcriptional activator in the proximity of a promoter that controls gene transcription. Typically, a transcriptional activator is associated with a protein that has a DNA binding domain that binds to sites present in the promoter regions of genes. For gene expression to occur, a protein comprising a DNA binding domain and a transactivation domain must be brought into the correct position in the promoter region of a gene.
One transgenic approach utilizes a cell-type specific promoter to drive the expression of a transgene. A DNA construct containing the transgene is first incorporated into a host genome. When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.
Another approach is through inducible promoters. Examples include the PR1-a promoter, prokaryotic repressor-operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems.
Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc. Natl. Acad. Sci. USA 83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61; Filmus et al., 1992 Nucleic Acids Research 20:27550-27560). However, these systems are leaky and have limitations due to their effect on expression of non-target genes.
Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind. Both the tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression. In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change that releases the repressor protein from the operator which as a result allows transcription to occur. In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-β-D-thiogalactoside. The use of such systems in plants and animals is restricted by unstable chemistry of the ligands (tetracycline and lactose), their toxicity, their natural presence, or the relatively high levels required for induction or repression.
Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc. Using this information, a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one. A synthetic homodimer of FK506 (FK1012) or a compound resulting from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993, Science 262:1019-24; Belshaw et al., 1996 Proc Natl Acad Sci USA 93:4604-7). Gal4 DNA-binding domain fused to FKBP12 and VP16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4-binding sites. This system includes immunosuppressants that can have unwanted side effects resulting in limited utility in mammalian gene switch applications.
Transcription activation systems such as steroid hormone receptor systems have also been employed. Steroid hormone receptors are members of a nuclear receptor superfamily and are found in vertebrate and invertebrate cells.
Growth, molting, and development in insects are regulated by the ecdysteroid hormones (molting hormones) and the juvenile hormones (Dhadialla, et al., 1998. Annu. Rev. Entomol. 43: 545-569). The molecular target for ecdysteroids in insects include ecdysteroid receptor (EcR) and ultraspiracle protein (USP). EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al. 1991, Cell, 67:59-77). EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A, as well as non-steroidal compounds including commercially available tebufenozide and methoxyfenozide (see PCT/EP96/00686 and U.S. Pat. No. 5,530,028).
The insect ecdysteroid receptor (EcR) heterodimerizes with Ultraspiracle (USP, the insect homologue of the mammalian RXR), binds ecdysteroids, binds ecdysteroid receptor DNA response elements, and activates transcription of ecdysteroid responsive genes. The EcR/USP/ligand complexes play important roles during insect development and reproduction. The EcR is a member of the steroid hormone receptor superfamily and has five modular domains, A/B (transactivation), C (DNA binding, heterodimerization), D (Hinge, heterodimerization), E (ligand binding, heterodimerization and transactivation and F (transactivation) domains. Some of these domains such as A/B, C and E retain their function when they are fused to other proteins.
Tightly regulated inducible gene expression systems or “gene switches” are useful for various applications such as gene therapy, large scale production of proteins in cells, cell based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.
A version of EcR-based gene switch used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptors in the presence of steroid, ponasterone A, transactivate reporter genes in mammalian cell lines and transgenic mice (Christopherson K. S., Mark M. R., Baja J. V., Godowski P. J. 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 6314-6318; No D., Yao T. P., Evans R. M., 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 3346-3351). Later, Suhr et al. 1998, Proc. Natl. Acad. Sci. 95:7999-8004 showed that tebufenozide induced transactivation of reporter genes in mammalian cells through Bombyx mori EcR (BmEcR) in the absence of exogenous heterodimer partner.
PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysteroid response element is activated by an ecdysteroid receptor that in the presence of a ligand and optionally in the presence of a receptor capable of acting as a silent partner. The ecdysteroid receptor was isolated from Drosophila melanogaster. Typically, such systems require the presence of the silent partner, such as retinoid X receptor (RXR), in order to provide optimum activation. In mammalian cells, insect ecdysteroid receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand-dependent manner. PCT/US98/14215 (WO 99/02683) discloses that the ecdysteroid receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.
U.S. Pat. No. 6,265,173 B1 discloses that various members of the steroid/thyroid superfamily of receptors can combine with Drosophila melanogaster USP or fragments thereof comprising at least the dimerization domain of USP for use in a gene expression system. U.S. Pat. No. 5,880,333 discloses a Drosophila melanogaster EcR and USP heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins. These USP-based systems are constitutive in animal cells and therefore are not effective for regulating expression of a gene of interest.
In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in PCT/US98/14215 or as modified EcR as in PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state.
Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat. No. 5,880,333).
Therefore, a need exists in the art for improved EcR-based systems to precisely modulate the expression of endogenous or exogenous genes of interest in both plants and animals. Such improved systems would be useful for applications such as gene therapy, large-scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic animals. For certain applications such as gene therapy, it may be desirable to have an inducible gene expression system that responds well to non-steroidal ligands and is insensitive to steroids, e.g., endogenous steroids. Thus, improved systems that are simple, compact, and dependent on ligands that are relatively inexpensive, readily available, and of low toxicity to the host are useful for regulating biological systems.
It has been shown that a nuclear receptor-based inducible gene expression system in which the transactivation and DNA binding domains are separated from each other by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand (PCT/US01/09050). This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the systems disclosed in applications PCT/US97/05330 and PCT/US98/14215. The two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Pat. No. 5,283,173). Briefly, the two-hybrid gene expression system comprises two gene expression cassettes; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide, and the second encoding a transactivation domain fused to a different nuclear receptor polypeptide. In the presence of ligand, the interaction of the first polypeptide with the second polypeptide effectively tethers the DNA binding domain to the transactivation domain. Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.
Furthermore, the two-hybrid system avoids some side effects due to overexpression of RXR that often occur when unmodified RXR is used as a switch partner. In one example of a two-hybrid system, native DNA binding and transactivation domains of EcR or RXR are eliminated. As a result, these hybrid molecules have lower interaction with other steroid hormone receptors present in the cell resulting in reduced side effects.
With the improvement in receptor-based gene regulation systems there is increased demand for ligands with higher activity than existing ligands. Disclosed herein are novel steroidal ligands which have the ability to modulate the expression of transgenes. See Silvia Lapenna Dissertation, United Kingdom.
Additional gene switch systems owned by applicant include those described in the following, each of which are incorporated by reference: U.S. Pat. No. 7,091,038; WO2004078924; EP1266015; US20010044151; US20020110861; US20020119521; US20040033600; US20040197861; US20040235097; US20060020146; US20040049437; US20040096942; US20050228016; US20050266457; US20060100416; WO2001/70816; WO2002/29075; WO2002/066612; WO2002/066613; WO2002/066614; WO2002/066615; WO2005/108617; U.S. Pat. No. 6,258,603; US20050209283; US20050228016; US20060020146; EP0965644; U.S. Pat. No. 7,304,162; U.S. Pat. No. 7,304,161.